We will characterize the mechanism by which the binding of cholinergic agonists and antagonists to the nicotinic acetylcholine receptor controls the permeability of the post-synaptic membrane. Torpedo electric tissue will be fractionated to isolate post-synaptic membranes. The permeability response will be measured by the flux of 22 Na ion and other isotopes from those membranes, and techniques will be used to measure a response directly proportional to the number of open ion channels. We will study the effects on the response of known concentrations of agonists and antagonists when present for times as short as 10 msec. Ionic environment at the internal and external surfaces of the vesicles will be varied in a systematic manner to determine the mechanisms that limit the number of ion channels that can be opened by agonist and the transport of ions through the nicotinic ion channel. The equilibrium and kinetics of binding of radiolabeled and fluorescent agonists and antagonists will be determined to define the binding properties of different receptor conformations, and the combined results of the binding and flux studies will be used to define the mechanism by which the binding of agonists results in the opening of the ion channel and then desensitization. Analysis of antagonist mechanisms will focus on a comparison of the actions of drugs that bind to the AcCh binding site (competitive antagonists) and those non-competitive antagonists that bind to the distinct anesthetic binding site in the Torpedo membranes. Studies will be carried out to define the relationship between that anesthetic binding site and the nicotinic ion channel. To study the role of lipid environment in the control of the nicotinic permeability response, nicotinic receptors will be isolated in detergent solution and reincorporated in artificial lipid vesicles of defined size and composition.